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Degradable and biocompatible nanoparticles decorated
with cyclic RGD peptide for efficient drug delivery to
hepatoma cells in vitro.
Pascal Loyer, Wahib Bedhouche, Zhi Wei Huang, Sandrine Cammas-Marion
To cite this version:
Pascal Loyer, Wahib Bedhouche, Zhi Wei Huang, Sandrine Cammas-Marion. Degradable and bio-compatible nanoparticles decorated with cyclic RGD peptide for efficient drug delivery to hep-atoma cells in vitro.. International Journal of Pharmaceutics, Elsevier, 2013, 454 (2), pp.727-37. �10.1016/j.ijpharm.2013.05.060�. �hal-00861299�
1
Degradable and Biocompatible Nanoparticles Decorated with Cyclic RGD Peptide for Efficient
1
Drug Delivery to Hepatoma Cells In Vitro
2
Pascal Loyer1, Wahib Bedhouche1, Zhi Wei Huang2,§, Sandrine Cammas-Marion2,*. 3
1
Inserm UMR S-991, Foie, Métabolismes et Cancer; Université de Rennes 1; Fédération de Recherche 4
de Rennes Biosit; CHU Rennes, 35033 Rennes, France. 5
2
UMR 6226 CNRS; Institut des Sciences Chimiques de Rennes; Université de Rennes 1; ENSCR, 6
Avenue du Général Leclerc, CS 50837, 35 708 Rennes Cedex 7, France. 7
§
Current address: UMR 6270 CNRS; Laboratoire Polymères Biopolymères Surfaces; Université de 8
Rouen, 76821 Mont Saint Aignan, France. 9
* Author to whom correspondence should be addressed
10
e-mail: sandrine.marion.1@ensc-rennes.fr; Tel: +33 2 23 23 81 09; Fax: +33 2 23 23 80 46. 11
12
Keywords: Biotinylated nanoparticles; site-specific targeting; anti-cancer drug encapsulation; 13
degradable poly(benzyl malate) derivatives; cyclic RGD peptide; HepaRG hepatoma cells. 14
15
ABSTRACT 16
Amphiphilic derivatives of poly(benzyl malate) were synthesized and characterized with the aim of 17
being used as degradable and biocompatible building blocks for the design of functional nanoparticles 18
(NPs). An anti-cancer model drug, doxorubicin, has been successfully encapsulated into the prepared 19
NPs and its release profile has been evaluated in water and in culture medium. NPs bearing biotin 20
molecules were prepared either for site-specific drug delivery via the targeting of biotin receptors 21
overexpressed on the surface of several cancer cells, or for grafting biotinylated cyclic RGD peptide 22
onto their surface using the strong and highly specific interactions between biotin and the streptavidin 23
protein. We have shown that this binding did not affect dramatically the physico-chemical properties 24
of the corresponding NPs. Cyclic RGD grafted fluorescent NPs were more efficiently uptaken by the 25
2 HepaRG hepatoma cells than biotinylated fluorescent NPs. Furthermore, the targeting of HepaRG 26
hepatoma cells with NPs bearing cyclic RGD was very efficient and much weaker for HeLa and HT29 27
cell lines confirming that cyclic RGD is a suitable targeting agent for liver cells. Our results also 28
provide a new mean for rapid screening of short hepatotropic peptides in order to design NPs showing 29
specific liver targeting properties. 30
31
1. Introduction
32
Nanotechnology, especially nanomedicine corresponding to the use of nanoparticles (NPs) in 33
biomedicine, is currently an ever growing scientific and technological domain [Psimidas et al., 2012; 34
Garanger et al., 2012]. The main reason for this unprecedented development relies on the aim to 35
improve both early detection and treatment of numerous pathologies such as cancers. The 36
encapsulation of a selected biologically active molecule into NPs might result in an increased drug 37
bioavaibility within solid tumors, arising from a decrease in its non-specific recognition by the 38
reticuloendothelial system (RES) and an improvement of its in vivo specific biodistribution, as well as 39
a minimized toxicity against healthy tissues and organs [Yan et al., 2012; Elsaesser et al., 2012; 40
Lamprecht, 2008]. Knowing that cancer, characterized by an abnormal and anarchical cell proliferation 41
within normal tissue, is a very complex disease and a major cause of mortality [Misra et al., 2010], the 42
development of efficient nanomedicine is thus a major challenge for public health [Reddy et al., 2011]. 43
In this context, several anti-cancer drug loaded NPs such as Doxyl® and Abraxane® have been 44
approved by the Food and Drug Administration (FDA) for clinical uses [Yan et al., 2012; Wang et al., 45
2012; Jain et al., 2010]. However besides these encouraging results, several challenges have to be 46
overcome in order to obtain NPs allowing highly efficient site-specific drug delivery. 47
The materials constituting the NPs have to respect very strict specifications: they must be (i) 48
biocompatible and non-toxic, (ii) (bio)degradable into non-toxic low molecular weight molecules or, at 49
least, bioassimilable after releasing the encapsulated drug, (iii) undetectable by the RES meaning 50
having stealth properties, (iv) adapted for carrying large amounts of drug that should be released in a 51
3 controlled manner at its site of action (targeting). Within this context, we have recently developed a 52
family of degradable non-toxic polymers derived from poly(malic acid), PMLA, which are able to 53
form well-defined NPs [Huang et al., 2012]. We have selected PMLA as macromolecular backbone 54
because this polymer, originally synthesized for application in the biomedical field, has been 55
successfully used as a platform in the synthesis of nanovectors [Huang et al., 2012; Cammas et al., 56
2000; Cammas-Marion et al., 2000; Osanai et al., 2000; Martinez Barbosa et al., 2004; Abdellaoui et 57
al., 1998] and macromolecular conjugates [Ding et al., 2010; Ljubimova et al., 2008; Fujita et al., 58
2007; Fujita et al., 2006]. PMLA is known to be non-toxic and degradable into malic acid under 59
physiological conditions [Vert et al., 1979] and its derivatives are accessible from naturally occurring 60
PMLA [Ljubimova et al., 2008] or by anionic ring-opening polymerization (ROP) of β-substituted β -61
lactones [Cammas et al., 1996; Cammas et al., 1993]. PMLA derivatives used for the formulation of 62
NPs were obtained by ROP of benzyl malolactonate (MLABe) in presence of either 63
tetraethylammonium benzoate, α-methoxy ω-carboxy poly(ethylene glycol) -PEG42-CO2H- or α-biotin
64
ω-carboxy poly(ethylene glycol) -Biot-PEG62-CO2H- as initiator [Huang et al., 2012]. Starting from
65
these three PMLA derivatives, we were able to obtain well-defined non-toxic NPs in which the 66
doxorubicin (Dox), and a fluorescent probe, the DiD oil, have been successfully encapsulated for in 67
vitro assays [Huang et al., 2012]. It is worth noting that PEG has been selected as hydrophilic block 68
because it is a well-known polymer conferring stealth properties at nanoparticles on which it is grafted 69
[Romberg et al., 2008]. On the other hand, biotin has been chosen firstly because it is a targeting agent 70
of certain cancer cells [Le Droumaguet et al., 2012; Patil et al., 2099; Kim et al., 2007] and secondly 71
because it is able to interact strongly with streptavidin [Yang et al., 2009] which is an important 72
property for our study as it will be explained afterwards in this paper. 73
Hepatocellular carcinoma (HCC) is the main primary malignant tumor of the liver representing 80 to 74
90% of liver tumors. It is the fifth most common tumor worldwide (5.4% of new cancer cases per year) 75
and the third in term of mortality (8.2% of all cancer death) [Parkin et al., 2005]. Early detection and 76
classification of HCC are crucial for the choice and effectiveness of therapeutic strategy. For small size 77
4 HCC, surgical treatment (resection and liver transplantation) is the most effective treatment [Hasegawa 78
et al., 2009; Mazzeferro et al., 2008; Ishikawa et al., 1992]. Palliative treatments such as 79
chemoembolization [Bernades-Genisson et al., 2003] and, more recently, chemotherapy using an 80
inhibitor of tyrosine kinase, Sorafenib (Nexavar®) are proposed to patients with advanced HCC. 81
Despites these advances, the therapeutic options for the treatment of HCC remains limited partly due 82
to the chemoresistance of liver tumors to conventional anti-tumor agents. Therefore, the use of 83
nanocarriers containing anti-tumor drugs has been envisaged for the treatment of HCC in order to 84
increase the intra-hepatic drug concentration while limiting the exposure of healthy tissues and side 85
effects [Reddy et al., 2011]. Several formulations are currently undergoing clinical trials in phase II 86
and III such as NPs of poly(alkyl cyanoacrylate) loaded with doxorubicin (Trandrug®) for HCC 87
treatment [Barraud et al., 2005]. In a first step, the passive accumulation of nanocarriers in the RES 88
cells (endothelial and Kupffer cells) [Lanaerts et al., 1984] was utilized for liver targeting with a real 89
relevance for diseases involving liver Kupffer cells such as parasitic diseases [Alving et al., 1978]. 90
Conversely, liver targeting based on the nanocarrier’s uptake by Kupffer cells has the major drawback 91
of allowing only a low hepatic accumulation of nanocarriers since Kupffer cells represent only a few 92
percent of liver cell volume against 90% for hepatocytes. In addition, the accumulation of nanocarriers 93
in Kupffer cells does not target the cells responsible for HCC thus limiting the use of this approach in 94
this case. Therefore, to overcome this drawback, active hepatocyte targeting has been studied. Most of 95
the proposed strategies are based on the binding of NPs to the asialoglycoprotein receptors [Wu et al., 96
2002]. These NPs are usually liposomes incorporating glycosylated proteins or galactose/lactose linked 97
to lipophilic anchors [Wu et al., 2002]. To date, very few nanovectors, especially polymer-based NPs, 98
carrying peptides with high tropism for the liver have been developed [Reddy et al., 2011]. 99
In this paper, we report, first, the monitoring of Dox release from NPs prepared from PEG42
-b-100
PMLABe or Biot-PEG62-b-PMLABe in water and in culture medium at 37°C. Second, we have grafted
101
fluorescein amine (FA) molecule at the free end of the PMLABe block of PEG42-b-PMLABe or
Biot-102
PEG62-b-PMLABe block copolymers in order to obtain fluorescent NPs for in vitro cell uptake assays.
5 Starting from the corresponding fluorescent NPs, we have studied the influence of the nature of 104
molecules localized at NPs’ surfaces [PEG42, Biot-PEG62 or Arginine-Glycine-Aspartic acid (RGD)
105
peptide-Biot-Streptavidin-Biot-PEG62] on their internalization into cells in vitro and demonstrated that
106
the uptake by HepaRG hepatoma cells is considerably enhanced by the grafting of the RGD peptide 107
onto NPs. These results also show that these biotinylated NPs can be useful tools for rapid in vitro 108
screen and selection of highly hepatotropic peptides with the ultimate goal to design nanomedicine 109
targeting hepatocytes from HCC in vivo. 110
111
2. Materials and methods
112
2.1. Materials 113
All chemicals were used as received. Anhydrous THF was obtained by distillation over 114
sodium/benzophenone under N2 atmosphere.
115
Three cell lines have been selected within the frame of this project: the HepaRG hepatoma cells 116
[Gripon at al., 2002; Laurent et al., 2010;], the colorectal adenocarcinoma cell line HT29 [Fogh et al., 117
1975] and the cervical cancer cell line HeLa [Rahbari et al., 2009]. 118
119
2.2. Apparatus 120
Nuclear magnetic resonance spectra (1H NMR) were recorded on a Brucker ARX 400 instrument (1H 121
at 400 MHz). Data are reported as follows: chemical shift (multiplicity, number of hydrogen). The 122
chemical shifts (δ) are reported as parts per million (ppm) referenced to the appropriate residual solvent 123
peak. Abbreviations are as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of 124
doublet), m (multiplet). 125
The size (average diameter obtained by the cumulant result method), polydispersity and zeta potential 126
of the formulations were measured by dynamic light scattering using a Delsa™ Nano Beckman 127
Coulter apparatus at 25°C. 128
UV spectra were recorded on a Secoman apparatus at 485 nm. 129
6 2.3. Synthesis of PEG42-b-PMLABe and Biot-PEG62-b-PMLABe
130
The monomer, the benzyl malolactonate (MLABe), was synthesized from DL-aspartic acid according 131
to the previously reported synthesis [Cammas et al., 1996]. The PEG42-b-PMLABe and Biot-PEG62
-b-132
PMLABe block copolymers were obtained by anionic ring opening polymerization of MLABe in 133
presence of, respectively, α-methoxy ω-carboxy poly(ethylene glycol), PEG42-CO2H, and α-biotin ω
-134
carboxy poly(ethylene glycol), Biot-PEG62-CO2H, as initiators following a protocol described
135
elsewhere [Huang et al., 2012]. 136
137
2.4. Grafting of fluorescein amine (FA) 138
2.4.1 Grafting of FA on the PEG42-b-PMLABe block copolymer (Scheme 1)
139
The PEG42-b-PMLABe block copolymer (500 mg) was dissolved into 1 mL of anhydrous CH2Cl2
140
under nitrogen atmosphere. To this solution were added 5 mg (1eq.) of N,N’-diclohexylcarbodiimide 141
(DCC) dissolved in 1 mL of anhydrous CH2Cl2 followed by 7.5 mg (1eq.) of N-hydroxysuccinimide
142
(NSH) dissolved in 1 mL of anhydrous CH2Cl2. The mixture was stirred under nitrogen atmosphere for
143
24 hours at room temperature (RT). The resulting PEG42-b-PMLABe-NHS was precipitated in cold
144
heptane. After the elimination of the supernatant, the precipitate was dissolved in CH2Cl2 and the
145
solution was filtrated on celite. The CH2Cl2 was eliminated under vacuum and the activated block
146
copolymer polymer (480 mg) was obtained with 96% yield. The activated block copolymer (480 mg) 147
was then dissolved in 1 mL of anhydrous CH2Cl2. To this solution was added 12 mg of FA (1eq.)
148
solubilised into a mixture of 1 mL of anhydrous CH2Cl2 and1 mL of acetone HPLC grade under
149
nitrogen atmosphere. After stirring at RT for 24 hours, the solution containing the PEG42
-b-PMLABe-150
FA block copolymer was precipitated into cold heptane. After removing the supernatant, the 151
precipitate was dissolved into DMSO. This DMSO solution was poured into a dialysis bag (MWCO 152
3,500 Da) and the dialysis was conducted during 8 hours against DMSO. The solution contained into 153
the dialysis bag was lyophilized and the PEG42-b-PMLABe-FA block copolymer was recovered with
154
60% yield. The polymer was characterized by 1H NMR in deuterated DMSO. 155
7 1 H NMR (d6-DMSO, δ ppm): 2.92 (s, 2nH, CO2CH2C6H5), 3.32 (m, 4mH (m=42), (CH2CH2O)42), 5.10 156 (m, 2nH, CHCH2CO2), 5.42 (m, 1nH, CHCH2CO2), 6.50-7.00 (m, 9H, FA); 7.31 (m, 5nH, 157 CO2CH2C6H5). 158
MNMR = 11,000 g/mol for the PMLABe block 159
160
2.4.2 Grafting of FA on the Biot-PEG62-b-PMLABe block copolymer (Scheme 1)
161
The grafting of FA on the Biot-PEG62-b-PMLABe block copolymer was realized as described above.
162
The Biot-PEG62-b-PMLABe-FA block copolymer was obtained with 58% yield and characterized by
163 1 H NMR in DMSO. 164 1H NMR (d6-DMSO, δ ppm): 2.92 (s, 2nH, CO 2CH2C6H5), 3.37 (m, 4mH (m=62), (CH2CH2O)62), 5.07 165 (m, 2nH, CHCH2CO2), 5.41 (m, 1nH, CHCH2CO2), 6.50-7.00 (m, 9H, FA); 7.26 (m, 5nH, 166
CO2CH2C6H5). Peaks corresponding to the biotin are either under peaks correspoonding to the PEG 167
and PMLABe blocks or too small to be detectable on the NMR spectrum. 168
MNMR = 6,000 g/mol for the PMLABe block. 169
170
2.5. Preparation of NPs 171
2.5.1. Dox encapsulation and release from NPs 172
The protocol for Dox encapsulation and for the monitoring of its release was the same whatever the 173
nature of the block copolymer constituted the NPs. The encapsulation of Dox into the NPs was realised 174
as described previously [Huang et al., 2012]. Briefly, the Dox hydrochloride (Dox,HCl, Sigma) was 175
encapsulated into the two kinds of NPs during the nanoprecipitation procedure. The selected polymer 176
(5 mg) was dissolved in acetone (1 mL). Two hundred µ L of a Dox solution [1.5 mg of Dox,HCl 177
solubilised in 0.6 mL of a mixture of chloroform (6 mL) and NEt3 (23 µ L)] were added to the polymer
178
solution. This mixture was then nanoprecipitated into 2 mL of water under vigorous stirring. After 179
organic solvent evaporation, the unloaded Dox was removed by ultracentrifugation at 15,000 g at 15°C 180
for 7 min using filter with an exclusion limit 10,000 Da. The filters were returned and centrifuged for 1 181
8 min at 1,000 g at 15°C. The volume of the recovered solutions was completed to 2 mL with distilled 182
water in order to obtain a final concentration in NPs of 2.5 g/L. The concentration of loaded Dox was 183
evaluated by UV at 485 nm, as described elsewhere [Huang et al., 2012; Cammas et al., 1995]. Briefly, 184
200 µ L of Dox-loaded NPs were dissolved into 800 µ L of DMF and the resulting solutions were 185
analyzed by UV at 485 nm. The absorbance of Dox encapsulated into NPs was converted into a 186
concentration using a calibration curve and the encapsulation efficiency (e.e.) was calculated using the 187
following equation: 188
189 190
The characteristics of the Dox-loaded NPs (diameter, polydispersity index and zeta potential) were 191
measured using the Delsa™ Nano Beckman Coulter apparatus (Table 1). 192
The Dox release from both kinds of NPs were monitoring by dialysis in water and in culture medium 193
at 37°C. The protocol used in both cases was identical. Two mL of the Dox-loaded NPs solution were 194
placed into a dialysis bag (MWCO = 3,500 Da); this bag was then incubated into 40 mL of water or 195
culture medium maintained at 37°C. After different incubation time, from 30 min up to 72 hours, 2 mL 196
of the outside solution are taken and replaced by 2 mL of fresh water or culture medium. For each 197
sample, 200 µ L of the collected outside solution were analyzed by UV at 485 nm and the quantity of 198
Dox was determined thanks to calibration curves previously realized in water and in culture medium 199 by UV measurements at 485 nm. 200 201 2.5.2. Preparation of fluorescent NPs 202
The fluorescent NPs were prepared by the nanoprecipitation technique as previously described 203
[Thioune et al., 1997; Huang et al., 2012]. Briefly, the mixture of PEG42-b-PMLABe-FA (2.5 mg) and
204
PEG42-b-PMLABe (2.5 mg) or Biot-PEG62-b-PMLABe-FA (2.5 mg) and Biot-PEG62-b-PMLABe (2.5
205
mg) were dissolved in 1 mL of acetone. This solution is added to 2 mL of water under vigorous 206
stirring. The organic solvent (acetone) was then evaporated under vacuum and the final volume was 207
9 completed to 2 mL with fresh water. The final concentration in block copolymers under NPs’ form was 208
2.5 g/L. The solutions containing the fluorescent NPs were characterized by dynamic light scattering 209
using a Delsa™ Nano Beckman Coulter apparatus at 25°C (Table 2). 210
211
2.5.3. Grafting of the cyclic RGD peptide 212
The selected biotinylated peptide, the cyclic Biot-RGD peptide (Eurogentec, Belgium), was grafted 213
onto the biotinylated NP’s surfaces via the streptavidin (Strept, AnaSpect, Eurogentec, Belgium) 214
protein. An aqueous solution of Biot-RGD peptide was prepared at a final concentration of 2.9 mM. In 215
parallel, an aqueous solution of streptavidin was also prepared with a final concentration of 178 µM. 216
The Biot-RGD peptide (2.7 µ L) and the streptavidin (22 µ L) solutions were mixed in a final volume of 217
50 µ L (H2O qsp) and incubated for 1 hour at 4°C. Then, the Biot-PEG62-b-PMLABe-FA or
Biot-218
PEG62-b-PMLABe based NP’s solution (35 µ L), previously prepared, was added to a final volume of
219
100 µ L (H2O qsp). This mixture was incubated for 1 hour and diluted to a final volume of 1 mL in
220
culture medium for final concentrations of the Biot-RGD peptide at 8µM, the streptavidin at 4µM and 221
polymers at 4µM. In order to demonstrate that both the Biot-RGD peptide grafting and the dilution 222
have no influence on NP’s characteristics, we analyzed the NPs formed in the conditions described 223
above by DLS (Table 3). 224
225
2.6. Cell uptake assays 226
The cell lines, HT29 [Fogh et al., 1975] and HeLa [Rahbari et al., 2009], were cultured as described in 227
the literature. The HepaRG cell line was cultured in the medium William’s E (Lonza) supplemented 228
with 2mM of glutamine (Gibco), 5 mg/L of insulin (Sigma), 10-5 M hydrocortisone hemisuccinate and 229
10% of fetal calf serum (Lonza) [Gripon at al., 2002; Laurent et al., 2010]. During the sub-culturing, 230
2.106 cells were seeded in a 75 cm3 flask. The medium was renewed every 48 hours. The sub-culturing 231
was realized by trypsinization every 2 weeks in order to maintain the progenitor phenotype. For an 232
10 optimal differentiation, the cells were maintained at confluence after the two weeks and the medium 233
was supplemented with 2% of dimethylsulfoxide (DMSO) [Laurent et al., 2013]. 234
For the cell uptake assays, the 24 wells culture plates were seeded with the selected cell line (HepaRG, 235
HT29 or HeLa) with 105 cells per well. Then the NPs’ preparations (4µM of block copolymer under 236
NPs’ form ± 4 µM streptavidin and 8 µM Biot-RGD) or a negative control (buffer without NPs or non 237
fluorescent NPs) were added to the wells. For the competitive experiments, the cells were pre-treated 238
with an excess of free RGD peptide (32 µM). 239
The cells were incubated from 1 to 24 hours. After incubation, the culture medium was removed; the 240
cell monolayers were washed with PBS before the observation by fluorescence microscopy (Zeiss 241
inverted microscope, analysis software AxioVision). Then the cells were detached with trypsin and 242
analyzed by flow cytometry (FACSCalibur Becton Dikinson) to quantify the fluorescence (Channel 243
FL1H) emitted by the fluorescent NPs captured by the cells. Cytometry data were analyzed using 244
CellQuest software (Becton Dikinson). 245
246
3. Results and Discussion
247
In order to further characterize the PMLABe based NPs as drug nanocarriers for applications in 248
nanomedicine, we (i) studied the release of Dox in water and culture medium at 37°C, (ii) grafted a 249
fluorescent probe at the free end of the hydrophobic PMLABe block for studying in vitro cellular 250
uptake and (iii) evaluated the possibility to build a molecular scaffold by grafting the cyclic RGD-251
biotinylated peptide onto biotinylated NPs via the streptavidin as a bridging factor and determine the 252
impact of the RGD peptide addition on cell uptake. 253 254 Figure 1 255 256 257 258
11 3.1. Dox encapsulation and release from NPs
259
Both Dox-loaded PEG42-b-PMLABe and Biot-PEG62-b-PMLABe based NPs have been prepared by
260
the nanoprecipitation technique and have been characterized by DLS. Table 1 collects the results 261
obtained for PEG42-b-PMLABe and Biot-PEG62-b-PMLABe based NPs. Initial Dox content in both
262
NPs determined by UV at 485 nm as described previously [Huang et al., 2012] showed an 263
encapsulation efficiency ranging from 32 to 36%. 264
265
Table 1 266
267
The release of Dox from both PEG42-b-PMLABe and Biot-PEG62-b-PMLABe NPs in water and in
268
culture medium was realized at 37°C by dialysis. Aliquots of the external solution (outside the dialysis 269
bag) were collected after various incubation times and analyzed by UV at 485 nm and confirmed that 270
the Dox was encapsulated with an efficiency of nearly 35% (Table 1). As shown in figure 2, Dox 271
release profiles from both types of NPs in water (Figure 2, A) and in the culture medium (Figure 2, B) 272
are quite similar. 273
274
Figure 2 275
276
The release, expressed as a percentage of the total amount of encapsulated Dox, is nearly 5% after one 277
hour of incubation. This release accelerates after the second hour to reach 40% after 6 hours of 278
incubation in both water and culture medium. Then the release reaches a plateau around 55 to 60% 279
from 24 to 72 hours of incubation. The fast release of Dox within the first hours is probably due to the 280
Dox absorbed in the hydrophilic PEG corona. Indeed, Dox is known to be an amphiphilic molecule 281
which is therefore spread from the hydrophilic corona to the surface of the hydrophobic core of the 282
nanoparticles. However to conclude regarding the exact location of the Dox in the PEG-b-PMLABe 283
forming NPs, it will be necessary to realize further experiments such as X-ray measurements. 284
12 Nevertheless, these results are quite encouraging because we are able to encapsulate substantial 285
amount of this drug and the release profile is in agreement with the Dox amphiphilic nature. 286
287
3.2. Grafting of fluorescein amine and cellular uptake assays 288
Besides the use of biotin as a well-known targeting agent of cancer cells [Yang et al., 2009], we aimed 289
at developing a versatile procedure to screen for more specific targeting agents, especially short 290
peptides, towards transformed hepatocytes from hepatocellular carcinoma (HCC). 291
Our goal is to select peptides exhibiting a remarkably high tropism for the hepatocytes as targeting 292
agents and to graft them at the surface of PMLA derivatives-based NPs in order to achieve an 293
optimized uptake of NPs by the hepatocytes. In a first step, we wish to screen, rapidly and in a simple 294
manner without engaging more organic chemistry, a large number of peptides which potentially show 295
a strong hepatotropism to select the most efficient ones. For that purpose, we used the non-covalent 296
binding of selected peptides via the strong biotin-streptavidin affinity as shown by Figure 3 [Yang et 297 al., 2009]. 298 299 Figure 3 300 301
In a first step to determine whether such a molecular scaffold could allow the production of NPs and 302
could be used for cell uptake in vitro, we have selected biotinylated cyclic RDG (Arg-Gly-Asp- DTyr-303
Lys-Biotin) peptide, known for interacting with integrin proteins well expressed in the liver and even 304
more in tumors rich in extracellular matrix [Jiang et al., 2011]. This biotinylated RGD peptide has been 305
introduced after the formation of biotinylated NPs through streptavidin interactions [Yang et al., 2009]. 306
Moreover, in order to follow the in vitro cellular uptake of the NPs, we have synthesized fluorescein 307
amine grafted PEG42-b-PMLABe and Biot-PEG62-b-PMLABe block copolymers. As shown by scheme
308
1, the fluorescein amine (FA) was successfully grafted at the free carboxylic acid end of the PMLABe 309
13 block activated with N-hydroxysuccinimide (NHS) without modifying the structure of block 310 copolymers. 311 312 Scheme 1 313 314
After purification by dialysis allowing the elimination of unreacted FA and low molecular weight side 315
products, both block copolymers were characterized by 1H NMR in deuterated DMSO. The 1H NMR 316
spectra allowed us to conclude that the structures of FA-modified block copolymers were in agreement 317
with the expected ones and that the molecular weights of PMLABe blocks calculated from the 1H 318
NMR spectra were identical to the ones of initial materials. 319
Starting from a mixture of the fluorescent block copolymers and the non-fluorescent ones (50/50 wt%), 320
we have then prepared the corresponding fluorescent NPs using the nanoprecipitation method [Huang 321
et al., 2012; Thioune et al., 1997]. The obtained NPs were characterized by dynamic light scattering. 322
As shown by results gathered in table 2, the presence of FA molecules at the end of the PMLABe 323
block has no significant influence on the NP’s diameters and polydispersity indices. 324
325
Table 2 326
327
Biotinylated cyclic RGD peptide was then associated with NPs formed by a mixture of Biot-PEG62
-b-328
PMLABe-FA and Biot-PEG62-b-PMLABe (50/50 wt%) using streptavidin, a tetrameric protein of 56
329
KDa purified from the bacterium Streptomyces avidinii, as an intermediate link between the biotin 330
localized on the surface of preformed polymeric NPs and the biotinylated peptide (Figure 3). The 331
dissociation constant (Kd) of the biotin/streptavidin complex is on the order of 10-15 mol/L, ranking 332
among the strongest known non-covalent interactions [Yang et al., 2009]. The non-covalent binding of 333
RGD modified fluorescent NPs was realized by first mixing the biotinylated RGD peptide with the 334
streptavidin followed by the addition of this complex to biotinylated fluorescent NPs. The relative 335
14 amounts of block copolymers constituting the NPs, RGD peptide and streptavidin can have a 336
significant influence on the cell capture. Therefore, different amounts of block copolymer constituting 337
the NPs, RGD peptide and streptavidin were tested and the cell uptake was measured by fluorescent 338
microscopy and flow cytometry (Data not shown); the best results were obtained with the following 339
conditions: 4 µM of block copolymers under NP’s form, 4 µM of streptavidin and 8 µM of 340
biotinylated RGD peptide. 341
The diameter and polydispersity index of unmodified and modified NPs were measured by DLS in 342
order to demonstrate that the addition of RGD-Biot-streptavidin construct and the dilution had no 343
influence on the properties of the corresponding NPs. As shown by results gathered in table 3, the 344
dilution of Biot-PEG62-b-PMLABe NPs either in PBS or in culture medium with or without serum
345
does not have a significant influence on both the diameter and the polydispersity index values, 346
meaning that the polymers constituting the NPs are still associated under NP’s form. 347
348
Table 3 349
350
The addition of the Strep-Biot-RGD construct on the biotinylated NPs has led to a moderated increase 351
in the NPs’ diameter and polydispersity indices. For NPs resuspended either in PBS or culture medium 352
with serum the emergence of a second peak centered at 600 nm and 970 nm, respectively, was 353
observed. Such results indicate that addition of Strep-Biot-RGD construct does not lead to the 354
destabilization of the NPs but rather a moderate formation of aggregates. However, such limited 355
modifications were considered acceptable for cell uptake in vitro assays. It is important to note that 356
RGD modified NPs were stable for at least 48 hours when stored at room temperature as shown by 357
values of diameters and polydispersity indices unchanged (measures realized by DLS, data not shown). 358
We then realized in vitro assays of cell captation using the human hepatoma HepaRG cell line [Laurent 359
et al., 2010; Gripon et al., 2002]. HepaRG cells were incubated for 24 hours in the presence of 360
biotinylated NPs modified or not with the RGD-Biot-Streptavidin construct with final concentrations 361
15 in culture media of 4 µM of block copolymers, 4 µM of streptavidin and 8 µM of RGD peptide. The 362
cell uptake was studied by Facs and fluorescent microscopy (Figure 4). 363
364
Figure 4 365
366
The Facs analysis showed that the peak of fluorescence in cells incubated with the Biot-PEG62
-b-367
PMLABe-FA NPs had significantly shifted on the right compared to fluorescence in control cells 368
incubated with non fluorescent NPs demonstrating that most of the cells contained fluorescent NPs. 369
However, the mean of fluorescence remained low (~30 arbitrary units) for Biot-PEG62-b-PMLABe
370
NPs and Biot-PEG62-b-PMLABe-FA NPs, respectively, compared to the mean for cells incubated with
371
non-fluorescent NPs. The fluorescent microscopy confirmed that HepaRG cell uptake of Biot-PEG62
-372
b-PMLABe-FA NPs was very limited compared to negative control cells exposed to the non-373
fluorescent NPs demonstrating that the biotin did not trigger a strong captation. Addition of the RGD 374
peptide led to strong increase in the fluorescence level within HepaRG cells reaching a mean of ~1500 375
U.A and with over 90% of positive cells (Figure 4). These data demonstrate that the addition of the 376
RGD peptide onto NPs has considerably enhanced the cell uptake of the NPs. 377
We then followed the time course of uptake by HepaRG cells of NPs formed by Biot-PEG62
-b-378
PMLABe-FA or Biot-RGD-Strept-Biot-PEG62-b-PMLABe-FA by flow cytometry analysis at 2, 6, 14
379
and 24 hours of incubation. As shown by figure 5, the results of mean of fluorescence intensity 380
indicated a very rapid uptake within the first 6 hours which continued slower until 24 hours for RGD-381
Biot-Strept-Biot-PEG62-b-PMLABe-FA based NPs (Figure 5, red curve), while no change in the
382
fluorescence intensity has been observed for the uptake of Biot-PEG62-b-PMLABe-FA based NPs
383
(Figure 5, blue curve). 384
385
Figure 5 386
16 Furthermore, we have evaluated the specificity of the uptake of RGD modified fluorescent NPs by 388
HepaRG cells by comparing the efficiency of the uptake in absence or presence of an excess of free 389
RGD peptide at a concentration of 34 µM (Figure 6). The results of fluorescence intensity measured by 390
flow cytometry indicated that the pre-incubation of HepaRG cells with free RGD peptide before the 391
addition of RGD modified fluorescent NPs strongly inhibited the cell uptake. Therefore, we can 392
conclude that the presence of the RGD peptide at the NP’s surfaces is responsible for the increase in 393
HepaRG cell uptake of NPs. 394
395
Figure 6 396
397
In final experiment, we compared the efficiency of the uptake of RGD modified fluorescent NPs by 398
three different cell lines: the hepatoma HepaRG, the colon HT29 and the cervical HeLa cancer cells 399 (Figure 7). 400 401 Figure 7 402 403
The three cell lines showed low levels of fluorescence following incubation with Biot-PEG62
-b-404
PMLABe-FA NPs for 24 hours demonstrating that these biotinylated NPs are poorly uptaken by cells 405
from different tissue origin. Importantly, the three cell types exhibited very different uptake of the 406
RGD-Biot-Strept-Biot-PEG62- b-PMLABe-FA formed NPs. The number of positive HT29 cells is very
407
low (< 5%) while about 90% of HeLa cells captured the RGD modified fluorescent NPs (Figure 7). 408
However, the degree of uptake (fluorescence intensity) in HeLa cells is much lower than the one 409
observed for HepaRG cells (Figure 7). This result suggested that the number of membrane receptors 410
binding RGD peptide (integrin family members) on HeLa cells is sufficient to trigger the uptake of 411
NPs by most of the cells. However, the density of these receptors onto HeLa cells might be lower than 412
17 onto HepaRG cells leading to a higher degree of NPs captation by the hepatoma cells than the HeLa 413 cells. 414 415 4. Conclusions 416
In this study, we have demonstrated a rapid release of the Dox from PEG42-b-PMLABe and
Biot-417
PEG62-b-PMLABe NPs over the first 6 hours. However, the NPs, which are stable in water and culture
418
medium for several days, entrapped a fraction of the drug that is slowly released over several days. 419
Then, we characterized the cell uptake of PEG42-b-PMLABe and Biot-PEG62-b-PMLABe NPs using
420
fluorescein amine modified polymers. We demonstrated that biotinylated NPs modified with the cyclic 421
RGD peptide significantly increase the uptake by HepaRG cells in comparison to NPs formed by Biot-422
PEG62-b-PMLABe-FA without peptide. We have also proved that this capture was dependent on the
423
presence of the RGD peptide because the addition of an excess of free RGD peptide strongly inhibited 424
the uptake. In addition, our results indicate that the RGD peptide presents a real tropism for liver cells 425
since its uptake by HeLa and HT29 cells was much lower than the uptake by HepaRG cells. These 426
results should be complemented by a larger study including other liver and non-hepatic cell lines as 427
well as primary cells such as endothelial cells and normal hepatocytes. Our study also provides a proof 428
of concept for the use of the versatile molecular scaffold presented in Figure 3 in order to screen for 429
other short peptides targeting hepatocytes and HCC. Since we have shown that it was possible to use 430
the streptavidin to graft a biotinylated peptide onto the biotin present at the surface of Biot-PEG62
-b-431
PMLABe based NPs, we are evaluating a number of peptides without having to engage additional 432
chemistry. The most efficient peptides will be then grafted at the end of the hydrophilic PEG block to 433
avoid the use of immunogenic streptavidin for further in vivo biodistribution assays. 434
435
Acknowledgments
436
We would like to thank Denise Glaise for culturing the HepaRG cells. This work was supported by 437
Inserm, CNRS, the University of Rennes 1 (Défis émergents-2012) and les comités départementaux de 438
18 la Ligue contre le Cancer du GrandOuest: comités 29, 35 et 53. Z.H. H. thanks the Région Bretagne 439
and the European University of Bretagne (UEB) for a Ph.D. grant and a 4 months mobility fellowship, 440 respectively. 441 442 References 443
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25 Table 1. Characteristics of Dox-loaded NPs.
600 NPs Diameter (nm)a Polydispersity index (Ip)a Zeta potential (mV)a Encapsulation efficiency (%)b PEG42-b-PMLABe 66 ± 7 0.22 - 8 ± 2 36 ± 3 Biot-PEG62-b-PMLABe 74 ± 4 0.22 - 6 ± 1 32 ± 2
a. Measured by DLS (Delsa™ Nano Beckman Coulter); b. Measured by UV at 485 nm.
601 602
26 Table 2. Characteristics of fluorescent NPs measured by DLS (Delsa™ Nano Beckman Coulter). 603
NPs Diameter (nm) Polydispersity index
PEG42-b-PMLABe-FA + PEG42-b-PMLABe (50wt%) 64 ± 14 0.16
Biot-PEG62-b-PMLABe-FA + Biot-PEG62-b-PMLABe (50wt%) 70 ± 13 0.22
604 605 606 607 608 609 610 611 612 613 614 615 616 617
27 Table 3. Characteristics of NPs modified or not by the strep-Biot-RGD construct measured by DLS 618
(Delsa™ Nano Beckman Coulter). 619 Conditions NPs Biot-PEG62-b-PMLABe NPs Biot-PEG62-b-PMLABe + strep-Biot-RGD Diameter (nm) Ip Diameter (nm) Ip Initial (2.5 g/L) 53 ± 12 0.17 -- --
Solution in PBS (0.107 g/L) 79 ± 15 0.37 208 ± 30 (a) 0.35(a)
Solution in culture medium without serum (0.107 g/L) 78 ± 15 0.29 191 ± 21 0.28
Solution in culture medium with serum (0.107 g/L) 106 ± 30 0.15 185 ± 30 (b) 0.23(b)
(a). Presence of a second peak centered at 600 ± 100 nm; (b). Presence of a second peak centered at 620
970 ± 220 nm 621
28 Figure captions:
623
Figure 1. Uses of PMLA derivatives as building blocks for the design of versatile NPs. 624
625
Figure 2. Release profile of Dox in water (A) and in culture medium (B) at 37°C. 626
627
Figure 3. Schematic representation of the molecular scaffold following grafting of biotinylated peptide 628
on the biotinylated NPs via streptavidin as a bridging factor. 629
630
Figure 4. Flow cytometry (right) and in situ fluorescence microscopy (left) analysis of HepaRG cells 631
incubated with Biot-PEG62-b-PMLABe NPs, Biot-PEG62-b-PMLABe-FA NPs and
RGD-Biot-Strept-632
Biot-PEG62-b-PMLABe-FA NPs. For Facs analysis, size (FSC-H) and granularity (SSC-H) were
633
visualized to select the R1 gate corresponding to living cells. Detection on fluorescent cells in the R1 634
gate was performed using the FL1-H channel: negative cells incubated with Biot-PEG62-b-PMLABe
635
NPs were set in the M1 window and positive cells were detected in the M2 window. For cells 636
incubated with Biot-PEG62-b-PMLABe-FA and RGD-Biot-Strept-Biot-PEG62-b-PMLABe-FA NPs (1)
637
50 and (1) 90% of cells were fluorescent, respectively; white bar : 50 µm. 638
639
Figure 5. Time course of the uptake of NPs by HepaRG cells. Red curve: RGD-Biot-Strept-Biot-640
PEG62-b-PMLABe-FA NPs; Blue curve: Biot-PEG62-b-PMLABe-FA NPs; white bar : 50 µm.
641 642
Figure 6. Captation of fluorescent nanoparticles (24 hour incubation) by HepaRG cells in absence or in 643
presence of free RGD peptide; Flow cytometry (Left) and fluorescence microscopy (Rigth) analyses 644
with Biot-PEG62-b-PMLABe-FA NPs, RGD-Biot-Strept-Biot-PEG62-b-PMLABe-FA NPs and
RGD-645
Biot-Strept-Biot-PEG62-b-PMLABe-FA NPs + 34 µM of free RGD peptide. Bottom chart: Mean of
646
fluorescence under the various tested conditions. 647
29 Figure 7. Flow cytometry measurements of fluorescence in HepaRG, HT29 and HeLa cells incubated 648
with Biot-PEG62-b-PMLABe-FA or RGD-Biot-Strept-Biot-PEG62-b-PMLABe-FA formed NPs for 24
649
hours. Left chart: Number of positive cells; right chart: Mean of fluorescence. 650
30 Figure 1. 652 653 654 PEG42-b-PMLABe Biot-PEG62-b-PMLABe PEG42-b-PMLABe-FA Biot-PEG62-b-PMLABe-FA Nanoprecipitation Dox ( )
PEG42-b-PMLABe [Dox] NPs
Biot-PEG62-b-PMLABe[Dox] NPs
Dialysis against H2O
or culture medium Dox release profile
Nanoprecipitation PEG42-b-PMLABe-FA NPs Biot-PEG62-b-PMLABe-FA NPs RGD-Biot-Strept-Biot-PEG62-b-PMLABe NPs RGD-Biot-Strept NPs’ uptake in vitro Biotin Streptavidin (Strept) RGD peptide
31 Figure 2. 655 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 10 20 30 40 50 60 70 80 R e le a se r a te ( % )
Incubation time (hours)
B PEG-b-PMLABe[Dox] NPs Biot-PEG-b-PMLABe[Dox] NPs 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 10 20 30 40 50 60 70 80 R e le a se r a te ( % )
Incubation time (hours)
A
PEG-b-PMLABe[Dox] NPs Biot-PEG-b-PMLABe[Dox] NPs
656 657
32 Figure 3. 658 659 660 661 662 663 664 665 123 4567 879AB7CD5E5F 2AB75EA AAB769 A 9
33 Figure 4.
666
667 668
HepaRG control neg.001
0 200 400 600 800 1000 FSC-H R1 HepaRG PMLAbiotFA.002 0 200 400 600 800 1000 FSC-H R1 HepaRG PMLAbiotFARGD c3.003 0 200 400 600 800 1000 FSC-H R1 Biot-PEG62-b-PMLABe NPs E v e n ts E v e n ts E v e n ts S S C -H S S C -H S S C -H Biot-PEG62-b-PMLABe-FA NPs RGD-Biot-PEG62-b-PMLABe-FA NPs HepaRG control neg.001
100 101 102 103 104 FL1-H M1 M2 HepaRG PMLAbiotFA.002 100 101 102 103 104 FL1-H M1 M2 100 101 102 103 104 FL1-H M1 M2
34 Figure 5. 669 670 671 672 673 674 675 676 677 0 50 100 150 200 250 300 350 400 0 6 12 18 24 Biot-PEG62-b-PMLABe-FA NPs RGD-Biot-PEG62-b-PMLABe-FA NPs Time (hours) F lu o re sc e n c e i n te n si ty ( A U )
35 Figure 6. 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 100 101 102 103 104 FL1-H M1 M2
HepRG PMLAFAbiot RGDcond3c.009
100 101 102 103 104 FL1-H M1 M2 100 101 102 103 104 FL1-H M1 M2 Biot-PEG62-b-PMLABe-FA NPs RGD-Biot-PEG62-b-PMLABe-FA NPs
RGD-Biot-PEG62-b-PMLABe-FA NPs + free RGD
E v e n ts E v e n ts E v e n ts 1 211 311 411 511 611 711 811 2 3 4 5 M e a n o f fl u o re s c e n c e ( U .A .)
36 Figure 7. 704 705 706 100 101 102 103 104 FL1-H M1 M2 100 101 102 103 104 FL1-H M1 M2
HeLa PMLA biotFA RGDc3.009
100 101 102 103 104 FL1-H M1 M2 1 31 51 71 91 211 2 3 4 5 6 7 8 9 A 1 211 311 411 511 611 2 3 4 5 6 7 8 9 A Me a n o f fl u o re s c e n c e ( U .A .) P o s it iv e c e ll s ( % )
HepaRG HT29 HeLa HepaRG HT29 HeLa
HeLa
HepaRG neg cond 1a.001
100 101 102 103 104 FL1-H
M1
M2
HepaRG PMLAFAbiot cond 2a.004
100 101 102 103 104 FL1-H
M1
M2
HepRG PMLAFAbiot RGDcond3c.009
100 101 102 103 104 FL1-H M1 M2 HepaRG Without NPs 100 101 102 103 104 FL1-H M1 M2 HT29 PMLA FA RGD c3.015 100 101 102 103 104 FL1-H M1 M2 HT29 PMLA FAc2.014 100 101 102 103 104 FL1-H M1 M2 HT29
37 Scheme captions
707 708
Scheme 1. Synthetic route to FA grafted PEG42-b-PMLABe and Biot-PEG62-b-PMLABe. 709
38 Scheme 1. 711 712 R b CH CH2 CO2CH2C6H5 C O O H n R: PEG42-; PEG42-b-PMLABe
R: Biot-PEG62-; Biot-PEG62-b-PMLABe
DCC/NHS CH2Cl2anhydrous RT, 24 h R b CH CH2 CO2CH2C6H5 C O O NSH n FA CH2Cl2anhydrous RT, 24 h R b CH CH2 CO2CH2C6H5 C O O FA n R: PEG42-; PEG42-b-PMLABe-FA
R: Biot-PEG62-; Biot-PEG62-b-PMLABe-FA O O OH HO H2N O Fluorescein amine, FA
